Research Summary The objectives of this project are to elucidate an unexplored metabonegenic regulation of citrate for bone development, and to translate these understandings towards the design of novel biomimetic citrate-presenting bone biomaterials for orthopedic applications. Although significant progress has been made in the development of orthopedic biomaterials, the currently available materials are limited by their inabilities to mimic the native tissue composition, weak mechanical strength, minimal osteoinductivity, significant inflammatory responses, poor bone integration, and slow bone regeneration. We hypothesize that the uptake of extracellular citrate via transporter SLC13a5 could elevate cellular energy status through modulation of cell metabolism, which in turn leads to a facilitated osteogenic phenotype progression by inhibiting the activity of AMP-activated protein kinase (AMPK). This new citrate-based regulation of bone development is referred to as citrate metabonegenic regulation (Fig. 1). The identification of new and unexplored citrate-based strategies to promote osteogenic differentiation of mesenchymal stem cell can be harnessed to more efficiently design the next generation of biomimetic orthopedic biomaterials to address the limitations of the previous materials. To test our hypotheses and achieve the objectives of this project, three aims are proposed: Aim 1) to elucidate the metabonegenic regulatory effect of citrate for MSCs osteogenic differentiation; Aim 2) to apply the understandings of the citrate molecular mechanism in the design of biomimetic citrate-presenting biomaterials to mediate MSCs differentiation; Aim 3) To evaluate the in vivo performance of anatomically and chemically mimetic citrate-presenting scaffolds in a rat critically sized cranial bone defect model. It is very intriguing that the unprecedented knowledge on the unexplored citrate mechanism will enable us to design the next generation of biomimetic dynamic orthopedic implants that may present citrate signals in demand during cellular and tissue development. The understanding on the citrate metabonegenic regulation for bone stem cell culture and dynamic bone materials design will not only advance the field of bone tissue engineering, but also profoundly impact a wide array of other conditions such as MSC adipogenic differentiation since MSCs is multipotent and the adipogenic differentiation also has high energy demand.